2.1. Clinical case report
The proband, a 29-year-old female from Fujian(Fig 1 ), China, who had primary amenorrhea, had been married for 5 years without conceiving and had been diagnosed with POI. She had a normal target height (160cm), and normal weight (55kg). Physical examination showed no dysmorphic features or breast development and normal intellectual development. ynaecological examination, it was clear that the patient had a sparse amount of pubic and armpit hair. A transvaginal ultrasound examination revealed that the bilateral ovaries were abnormally small,the left ovary was 1.23×1.00cm, and the right ovary was 1.55×0.74cm, but no obvious antral follicles were observed(Fig 2). Her basic hormone levels were as follows: FSH 62.52-78.60 IU/L, Luteinizing hormone (LH) 20.38-25.43 IU/L, estradiol (E2) 13.0-42.5 pmol/L, and anti-Müllerian hormone (AMH) 0.09 ng/ml. She had a normal 46,XX karyotype and FMR1 repeat lengths and a negative testing of adrenal cortical antibody. The biological parents of the proband were healthy and non-consanguineous. The proband’s biological mother and sister had normal menstrual histories, and the family did not report any history of systemic diseases or solid tumors. This study fully complied with the tenets of the Declaration of Helsinki and has been approved by the Ethics Board of the Women's and children's Hospital affiliated to Xiamen University, China. Informed consent was obtained from all participants before testing.
One hundred unrelated ethnically matched healthy female individuals (aged between 22 and 40, average age of 28) were recruited as controls, They were known to be menstruating regularly, had normal FSH levels(range, 2.5-10.1IU/L ; mean, 3.6± 1.9 IU/L) and normal pelvic ultrasound imaging.
2.2 Targeted exon capturing and next-generation sequencing
Total genomic DNA was extracted from peripheral blood leukocytes using the Blood Genomic DNA Mini Kit (Qiagen, Valencia, CA, USA). The concentration of DNA samples was analyzed using a NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA).The exomes were captured with SureSelect Human All Exon V6 (Agilent Technologies, Santa Clara, CA, USA) and sequenced on an average depth of 150x with the use of the Illumina HiSeq platform (Illumina, San Diego, CA, USA) according to the manufacturer's protocol.
2.3 Bioinformatic analysis
After Illumina HiSeq sequencing, raw NGS data were imported into FastQC software for assessing the quality and the high-quality reads were aligned to the human reference genome (GRCh37/hg19) using Burrows-Wheeler Aligner (BWA) software. Then, variant calling and annotation were performed using GATK software . Several databases, such as the Single Nucleotide Polymorphism Database (dbSNP)138, the 1000 Genome Project, the Exome Aggregation Consortium (ExAc), ClinVar, and the Genome Aggregation Database (gnomAD) were employed to select all variants with frequencies higher than 5%. In addition, online tools such as Human Splicing Finder，PolyPhen-2，and SIFT were applied to predict the potential effect on protein function.
2.4 Confirmation by Sanger sequencing
The mutations of the PSMC3IP gene were further confirmed by Sanger sequencing. The PCR products were sequenced on an ABI 3730xl DNA Analyzer (Applied Biosystems, Thermo Fisher Scientific). Sequencing results were analyzed using the DNASTAR Lasergene software (DNASTAR, Madison, WI, USA). Subsequently，the novel identified mutations were verified and screened in 100 unrelated healthy women (aged between 22 and 40, average age of 28) with DNA sequencing.